Functional Analysis of a Novel Genome-Wide Association Study Signal in SMAD3 That Confers Protection From Coronary Artery Disease

Abstract

Objective-A recent genome-wide association study meta-analysis identified an intronic single nucleotide polymorphism in SMAD3, rs56062135C>T, the minor allele (T) which associates with protection from coronary artery disease. Relevant to atherosclerosis, SMAD3 is a key contributor to transforming growth factor-β pathway signaling. Here, we seek to identify ≥1 causal coronary artery disease-associated single nucleotide polymorphisms at the SMAD3 locus and characterize mechanisms whereby the risk allele(s) contribute to coronary artery disease risk. Approach and Results-By genetic and epigenetic fine mapping, we identified a candidate causal single nucleotide polymorphism rs17293632C>T (D′, 0.97; r 2 , 0.94 with rs56062135) in intron 1 of SMAD3 with predicted functional effects. We show that the sequence encompassing rs17293632 acts as a strong enhancer in human arterial smooth muscle cells. The common allele (C) preserves an activator protein (AP)-1 site and enhancer function, whereas the protective (T) allele disrupts the AP-1 site and significantly reduces enhancer activity (P<0.001). Pharmacological inhibition of AP-1 activity upstream demonstrates that this allele-specific enhancer effect is AP-1 dependent (P<0.001). Chromatin immunoprecipitation experiments reveal binding of several AP-1 component proteins with preferential binding to the (C) allele. We show that rs17293632 is an expression quantitative trait locus for SMAD3 in blood and atherosclerotic plaque with reduced expression of SMAD3 in carriers of the protective allele. Finally, siRNA knockdown of SMAD3 in human arterial smooth muscle cells increases cell viability, consistent with an antiproliferative role. Phosphorylated SMAD3 then forms a complex with the common SMAD4 that subsequently translocates to the nucleus and regulates transcription. 6,7 Relevant to a role in atherosclerosis, in systems genetics analysis of multiple GWAS, we identified TGF-β signaling and SMAD transcriptional activities as enriched pathways for CAD association. 8 However, despite extensive data on the functions of TGF-β with respect to atherosclerosis, 9,10 the roles of SMAD proteins particularly SMAD3 and SMAD3 signaling are less well-understood. Conclusions-TheSMAD3 is expressed at low levels in healthy human aorta by immunohistochemistry and quantitative reverse transcription polymerase chain reaction (PCR).11 There is, however, a marked increase in SMAD3 and other SMAD proteins in fibrofatty lesions, with expression mostly limited to CD68-positive macrophages/macrophage-derived foam cells in these samples. Conversely, in fibrous atherosclerotic plaques, there are high levels of SMAD3 in vascular smooth muscle cells (SMCs), suggesting that the role of SMAD3 in atherosclerosis depends on cell type and stage of atherosclerosis.11 Higher SMAD3 expression in SMCs of the fibrous plaque coincides with TGF-β-mediated synthesis of collagen and other extracellular matrix proteins, which contribute to plaque stability.12 Rare SMAD3 mutations cause aneurysms-osteoarthritis syn...

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“…Other notable CAD-related functional studies include one by Turner et al, 23 where genetic and epigenetic fine-mapping were used to identify a causal SNP in intron 1 of SMAD3, rs17293632. Using chromatin immunoprecipitation and small interfering RNA-induced knockdowns, these investigators demonstrated that the rs17293632-T allele plays a protective role by disrupting a consensus AP (activator protein)-1 binding site in the SMAD enhancer region, reducing the activity of the enhancer and in turn, SMAD3 expression.…”